Gastroenteritis is used in the medical field to refer to patient infection or irritation of the digestive tract, particularly the stomach and intestine. Commonly, it may be referred to as “stomach flu” even though it is not related to influenza, or as “food poisoning.” Major symptoms include nausea and vomiting, diarrhea, and abdominal cramps, which can be accompanied by fever and overall weakness. While gastroenteritis does not normally pose significant threats to otherwise-healthy adults, children, the elderly, and persons with certain existing diseases are more vulnerable to dehydration and other complications.
Acute cases of gastroenteritis affect millions of persons per year in the U.S., and an estimated 22% to 30% of these cases are thought to be caused by food-borne disease or pathogens (i.e., “food poisoning”). For example, it has been reported that globally 1.3 billion cases of salmonellosis, food poisoning resulting from exposure to Salmonella enterica, occur annually, resulting in approximately 3 million deaths. While most otherwise healthy adults recover from such food poisoning within a few days of exposure, the symptoms can be at least temporarily debilitating. Because of the debilitating potential of acute gastroenteritis, bioterrorism through deliberate adulteration of a food supply using common, or, alternatively, more rare and deadly, pathogens poses a significant potential threat to national security. Given the wide variety of potential chemical and biological agents, contaminating food is perhaps one of the easiest means to intentionally introduce adulterants at many vulnerable points along the food supply continuum. Although all the diverse possibilities for food-borne bioterrorism cannot be specifically prevented, strategic preparations for surveillance, diagnosis, outbreak investigation, and medical response could mitigate foodborne threats of any origin.
A deliberate attack on the food supply is plausible and potentially catastrophic both economically as well as in loss of life. The major steps in meeting food defense goals include increasing preparedness, developing response plans and ensuring that we have tools to facilitate recovery. Principal among these tools would be a rapid, high-throughput screening technique to simultaneously detect and identify multiple food safety threat agents. Preferably, such tools should be capable of at least detecting selected bacterial agents having the potential for catastrophic public and economic consequences. Further, such tools should be able to provide reliable detection techniques to identify high-impact pathogenic agents in human food supply systems, before the agents reach the consumer.
Organisms listed in the national notifiable disease surveillance system and/or food-borne disease active surveillance system that have potential for use in bioterrorism include, for example, Escherichia coli O157:H7, Shigella dysenteriae, Salmonella enterica ssp. enterica (including serovars Typhi, Typhimurium, and Saintpaul) Francisella tularensis ssp. tularensis, Francisella tularensis ssp. novicida, Vibrio cholerae, Vibrio parahaemolyticus, Shigella sonnei, Yersinia pestis, and Yersinia pseudotuberculosis. The food threat agents Escherichia coli O157:H7, Salmonella, Shigella, Yersinia, and Vibrio cholerae have each been designated by the Centers for Disease Control and Prevention as high-impact food safety threat agents. Further, Francisella tularensis (henceforth, “F. tularensis”) is one of the most infectious pathogenic bacteria known, requiring inoculation or inhalation of as few as 10 organisms to cause extreme infectivity and having substantial capacity to cause illness and death. This feature and the capacity of the organism to survive outside of a mammalian host for weeks gives F. tularensis dangerous potential for use as a biological weapon (Kaufmann, Meltzer, Schmid, 1997; WHO, 2007).
Presently, there are no commercially viable mechanisms for wide and accurate screening and/or monitoring of the food supply for biothreat agents and food-borne pathogens. No single molecular diagnostic test is so far available to detect multiple food threats at once. Any viable mechanism would need not only to identify the presence of a potential agent with a high degree of sensitivity and accuracy, but also need to be able to identify exactly what agents are present. Preferably, any mechanism should be capable of identifying bio-threat agents from their closely related variants for the purpose of classifying and tracing the origin of contamination.
For example, because of their close evolutionary relationship, differentiation of Escherichia from Shigella species poses a big challenge (Jin et al., 2002; Pupo et al., 2000). Further, distinguishing within a genus, species, or subspecies can be just as challenging, such as in the case of distinguishing among Shigella sonnei from other members of its genus. In addition, certain organisms posing the potential to be food-borne bio-threat agents share extreme genome similarity with less threatening organisms, such as is the case with the highly virulent F. tularensis ssp. tularensis in comparison to the genetically similar, but less threatening to humans, F. tularensis ssp. mediastatica. In other instances, a complete genome sequence database is unavailable, making it difficult to localize specific regions within the genome, such as in the case of Salmonella enterica ssp. enterica serovar Saintpaul. The genomes of different strains of this serovar are not well characterized making it hard to develop molecular detection tools. Thus, the most common and/or concerning food threat bioorganisms are not presently easily detected and identified.
Additionally, any screening and monitoring mechanism must be simple to operate, and preferably should be able to detect and identify multiple target agents simultaneously. While research has considered multiplexed or simultaneous PCR-based molecular detection assays for food screening, heretofore there has not been successful adaptation of PCR technologies to food screening. One of the known challenges in multiplexed or simultaneous PCR-based molecular detections is the need for optimization of the reactions conditions such as annealing temperatures optimal for all primer sets, avoiding primer dimers, generation of compatible amplicon sizes, and adjustment for different amplification efficiencies (Edwards and Gibbs, 1994). Simply, adjusting the PCR for detecting one agent will oftentimes make it incompatible for simultaneous detection of another agent. This is especially complicated when the sample tested for contamination is a food product.
In particular, food matrices provide a critical challenge in amplification-based pathogen detection approaches. Because of potential spoilage, pre-analytical sample processing techniques are needed to reduce the time needed to arrive at diagnosis and decision-making (Benoit and Donahue, 2003; Dwivedi and Jaykus, 2011). Further, certain genetically-based detection mechanisms will not discriminate between live and dead organisms, with sterilized products containing non-viable bacteria or their DNA yield positive results on screening tests. Previous attempts have been made to develop multiplexed PCR assays that can simultaneously detect multiple food-borne pathogens (Fukushima et al., 2010; Jothikumar and Griffiths, 2002; Skottman et al., 2007; Wilson et al., 2005). None of those attempts were able to produce an assay that can detect and identify the primary food threat agents of bioterrorism potential, and none identified highly specific targets capable of discriminating a broad range of pathogens or related bacteria. Highly specific primer sequences are not available for all biothreat organisms of interest which primers are highly specific when tested against a wide array of organisms while also being suitable for simultaneous or multiplex detection of those organisms.
Thus, there remains a need in the art for methods, kits, and assays for the simultaneous, rapid and accurate detection and identification of multiple bio-threat agents that may be present in food.